Renal proximal tubular epithelial cells are significantly damaged during acute kidney injury. Renal proximal tubular cell-specific autophagy-deficient mice show increased sensitivity against renal injury, while showing few pathological defects under normal fed conditions. Considering that autophagy protects the proximal tubular cells from acute renal injury, it is reasonable to assume that autophagy contributes to the maintenance of renal tubular cells under normal fed conditions. To clarify this possibility, we generated a knock out mouse model which lacks Atg7, a key autophagosome forming enzyme, in renal proximal tubular cells (Atg7^flox/flox;KAP-Cre⁺). Analysis of renal tissue from two months old Atg7^flox/flox;KAP-Cre⁺ mouse revealed an accumulation of LC3, binding protein p62/sequestosome 1 (a selective substrate for autophagy), and more interestingly, Kim-1, a biomarker for early kidney injury, in the renal proximal tubular cells under normal fed conditions. TUNEL (TdT-mediated dUTP Nick End Labeling)-positive cells were also detected in the autophagy-deficient renal tubular cells. Analysis of renal tissue from Atg7^flox/flox;KAP-Cre⁺ mice at different age points showed that tubular cells positive for p62 and Kim-1 continually increase in number in an age-dependent manner. Ultrastructural analysis of tubular cells from Atg7^flox/flox;KAP-Cre⁺ revealed the presence of intracellular inclusions and abnormal structures. These results indicated that autophagy-deficiency in the renal proximal epithelial tubular cells leads to an increase in injured cells in the kidney even under normal fed conditions.

Cathepsin D is one of the major lysosomal aspartic proteases that is essential for the normal functioning of the autophagy-lysosomal system. In the kidney, cathepsin D is enriched in renal proximal tubular epithelial cells, and its levels increase during acute kidney injury. To investigate how cathepsin D-deficiency impacts renal proximal tubular cells, we employed a conditional knockout CtsD^flox/−; Spink3^Cre mouse. Immunohistochemical analyses using anti-cathepsin D antibody revealed that cathepsin D was significantly decreased in tubular epithelial cells of the cortico-medullary region, mainly in renal proximal tubular cells of this mouse. Cathepsin D-deficient renal proximal tubular cells showed an increase of microtubule-associated protein light chain 3 (LC3; a marker for autophagosome/autolysosome)-signals and an accumulation of abnormal autophagic structures. Renal ischemia/reperfusion injury resulted in an increase of early kidney injury marker, Kidney injury molecule 1 (Kim-1), in the cathepsin D-deficient renal tubular epithelial cells of the CtsD^flox/−; Spink3^Cre mouse. Inflammation marker was also increased in the cortico-medullary region of the CtsD^flox/−; Spink3^Cre mouse. Our results indicated that lack of cathepsin D in the renal tubular epithelial cells led to an increase of sensitivity against ischemia/reperfusion injury.

VPS13A is an intracellular lipid transfer protein comprising more than 3,000 amino acids. Mutations in human VPS13A cause VPS13A disease, a neurodegenerative disorder that affects movement and cognition. VPS13A forms a complex with the membrane protein XK to mediate ATP-induced phospholipid scrambling in the plasma membrane. Here, we established a mouse cell system expressing full-length mouse VPS13A and examined its interaction with XK. Mutational analysis revealed that VPS13A binds to XK through a C-terminal β-strand that interacts with a β-hairpin in the central region of XK, an interaction essential for scramblase activity. The XK paralog XKR2, which contains a similar β-hairpin structure, also associates with VPS13A and supports phospholipid scrambling. We analyzed 10 mouse VPS13A variants corresponding to human patient mutations and classified them into 4 groups: (a) L67P, I90K, and W2453R, which showed reduced expression; (b) A1091P and M3080R, which were normally expressed but lacked scramblase activity; (c) S1446P, Q2689H, Y2713C, and R3084H, which modestly impaired expression or activity; and (d) I2763R, which altered cell size and disrupted ER independently of XK. These findings define the VPS13A–XK interaction interface, clarify the functional impact of disease-causing mutations, and reveal an unexpected gain-of-function mutation of a VPS13A variant. In this study, we propose a model that explains how the neurodegenerative disease chorea-acanthocytosis develops.